Importance of pseudoaneurysms after TAVI - a retrospective analysis of 2063 patients.

Background: Bifemoral arterial access is common in patients undergoing transcatheter aortic valve implantation (TAVI), with a primary treatment access (TAVI access) and a secondary non-TAVI access. Pseudoaneurysm (PSA) is an important complication of femoral arterial puncture. Major vascular complications after TAVI are well described, but little is known about PSA. Patients and methods: A total of 2063 patients underwent transfemoral TAVI between January 2014 and January 2020. Vascular ultrasound of the common femoral artery was assessed before and after TAVI. We compared patient characteristics, periprocedural risk scores, procedural characteristics, and access site bleeding events according to Valve Academic Research Consortium 3 (VARC-3) criteria, length of stay (LOS), and all-cause mortality at one year between patients with (46) and without (2017) PSA. Results: The incidence of PSA after TAVI was 2.2% (46/2063). All PSA were successfully treated with ultrasound-guided manual compression (UGMC) or thrombin injection (UGTI) without complications. Patients with PSA had lower platelet counts (210×1000/µl vs. 234×1000/µl; p<0.05), more heart failure symptoms on admission (91% vs. 25%; p<0.05), were more often treated with (N)OACs for atrial fibrillation (AF; 54% vs. 38%; p <0.05), and were less often treated with aspirin (35% vs. 51%; p<0.03). Multivariate analysis identified secondary access site (odds ratio [OR] 8.11; p<0.001) and (N)OAC therapy (OR 1.31; p = 0.037) as risk factors for PSA development. PSA is associated with VARC-3 type 1-3 access site bleeding and longer LOS (15.2 ± 11.3 d vs. 11.6 ± 8.9 d; p<0.01), but this did not affect one year mortality (17% vs. 14%; p = 0.53). Conclusions: Pseudoaneurysms are an important complication after TAVI and are associated with access site bleeding and prolonged hospital stay. (N)OAC therapy and secondary access are important risk factors. Pseudoaneurysms can be safely and effectively treated with thrombin injection and do not affect one-year mortality.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.

Temporary decrease in microvascular tissue saturation after transcatheter aortic valve implantation.

BACKGROUND: Data on the effect of transcatheter aortic valve implantation (TAVI) on peripheral microcirculation are limited. OBJECTIVE: The aim of this study is to evaluate peripheral microvascular tissue saturation (StO2) before and after TAVI in relation to central and peripheral hemodynamics, cardiac and renal function. METHODS: In this single-center prospective study, patients with severe aortic stenosis (sAS) scheduled for TAVI or cardiac catheterization (control) were assessed before and up to five days after the procedure. Cardiac function including cardiac output (CO) was assessed by echocardiography. Brachial (bBP) and central blood pressure (cBP), ankle brachial index (ABI), and parameters of arterial stiffness, including augmentation pressure (AP) and augmentation index adjusted for heart rate (AIx@HR75) were measured to assess hemodynamic changes. StO2 was measured in all extremities using a near-infrared spectroscopy (NIRS) camera. Renal function was measured by creatinine levels. RESULTS: 26 patients underwent TAVI and 11 patients served as control. Cardiac output was significantly increased, whereas hemodynamic parameters and peripheral StO2 were significantly decreased after TAVI. At follow-up, StO2 returned to baseline values. Changes in StO2 were negatively related to creatinine levels. CONCLUSION: Transcatheter aortic valve implantation causes a temporary decrease in microvascular tissue saturation that is associated with renal function.

Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Divergent local and systemic antitumor response in primary uveal melanomas.

Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome 3 (M3) is associated with a high risk of metastases. M3 tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T cell infiltration correlates with better prognosis. Whether these T cells represent an antitumor response and how these T cells would be primed in the eye are both unknown. Herein, we characterized the T cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3 than in D3 tumors. CD39+PD-1+CD8+ T cells were enriched in M3 tumors, suggesting specific responses to tumor antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. scRNAseq-VDJ analysis of T cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions, suggesting in situ antitumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the antitumor responses in M3 and D3 tumors. Thus, tumor-Ag responses occur in localized UMs, raising the question of the priming mechanisms in the absence of known lymphatic drainage.

Familial uveal melanoma and other tumors in 25 families with monoallelic germline MBD4 variants.

BACKGROUND: Monoallelic germline MBD4 pathogenic variants were recently reported to cause a predisposition to uveal melanoma, associated with a specific tumor mutational signature and good response to immunotherapy. Monoallelic tumor pathogenic variants have also been described in brain tumors, breast cancers, and myxofibrosarcomas, whereas biallelic germline MBD4 pathogenic variants have been involved in a recessive hereditary adenomatous polyposis and a specific type of acute myeloid leukemia. METHODS: We analyzed MBD4 for all patients with a diagnosis of uveal melanoma at Institut Curie since July 2021 and in the 3240 consecutive female probands explored at the Institut Curie for suspicion of predisposition to breast cancer between July 2021 and February 2023. RESULTS: We describe 25 families whose probands carry a monoallelic germline pathogenic variant in MBD4. Eighteen of these families presented with uveal melanoma (including a case patient with multiple uveal melanoma), and 7 families presented with breast cancer. Family histories showed the first familial case of uveal melanoma in monoallelic MBD4 pathogenic variant carriers and other various types of cancers in relatives, especially breast, renal, and colorectal tumors. CONCLUSIONS: Monoallelic MBD4 pathogenic variant may explain some cases of familial and multiple uveal melanoma as well as various cancer types, expanding the tumor spectrum of this predisposition. Further genetic testing in relatives combined with molecular tumor analyses will help define the tumor spectrum and estimate each tumor's risk.

Mosaic BRCA1 promoter methylation contribution in hereditary breast/ovarian cancer pedigrees.

PURPOSE: Mosaic BRCA1 promoter methylation (BRCA1meth) increases the risk of early-onset breast cancer, triple-negative breast cancer and ovarian cancer. As mosaic BRCA1meth are believed to occur de novo, their role in family breast/ovarian cancer has not been assessed. PATIENTS: Blood-derived DNA from 20 unrelated affected cases from families with aggregation of breast/ovarian cancer, but with no germline pathogenic variants in BRCA1/2, PALB2 or RAD51C/D, were screened by methylation-sensitive high-resolution melting. CpG analysis was performed by pyrosequencing on blood and buccal swab. Two probands carried a pathogenic variant in a moderate-penetrance gene (ATM and BARD1), and 8 of 18 others (44%) carried BRCA1meth (vs none of the 20 age-matched controls). Involvement of BRCA1 in tumourigenesis in methylated probands was demonstrated in most tested cases by detection of a loss of heterozygosity and a homologous recombination deficiency signature. Among the eight methylated probands, two had relatives with breast cancer with detectable BRCA1meth in blood, including one with high methylation levels in two non-tumour tissues. CONCLUSIONS: The high prevalence of mosaic BRCA1meth in patients with breast/ovarian cancer with affected relatives, as well as this first description of a family aggregation of mosaic BRCA1meth, shows how this de novo event can contribute to hereditary breast/ovarian cancer pedigrees.

Male breast cancer: No evidence for mosaic BRCA1 promoter methylation involvement.

Breast cancers (BC) are rare in men and are often caused by constitutional predisposing factors. In women, mosaic BRCA1 promoter methylations (MBPM) are frequent events, detected in 4-8% of healthy subjects. This constitutional epimutation increases risk of early-onset and triple-negative BC. However, the role of MBPM in male BC predisposition has never been assessed. We screened 40 blood samples from men affected by BC, and performed extensive tumour analysis on MBPM-positive patients. We detected two patients carrying MBPM. Surprisingly, tumour analysis revealed that neither of these two male BCs were caused by the constitutional BRCA1 epimutations carried by the patients.

Shallow whole genome sequencing approach to detect Homologous Recombination Deficiency in the PAOLA-1/ENGOT-OV25 phase-III trial.

The bevacizumab (bev)/olaparib (ola) maintenance regimen was approved for BRCA1/2-mutated (BRCAmut) and Homologous Recombination Deficient (HRD) high-grade Advanced Ovarian Cancer (AOC) first line setting, based on a significantly improved progression-free survival (PFS) compared to bev alone in the PAOLA-1/ENGOT-ov25 trial (NCT02477644), where HRD was detected by MyChoice CDx PLUS test. The academic shallowHRDv2 test was developed based on shallow whole-genome sequencing as an alternative to MyChoice. Analytical and clinical validities of shallowHRDv2 as compared to MyChoice on 449 PAOLA-1 tumor samples are presented. The overall agreement between shallowHRDv2 and MyChoice was 94% (369/394). Less non-contributive tests were observed with shallowHRDv2 (15/449; 3%) than with MyChoice (51/449; 11%). Patients with HRD tumors according to shallowHRDv2 (including BRCAmut) showed a significantly prolonged PFS with bev+ola versus bev (median PFS: 65.7 versus 20.3 months, hazard ratio (HR): 0.36 [95% CI: 0.24-0.53]). This benefit was significant also for BRCA1/2 wild-type tumors (40.8 versus 19.5 months, HR: 0.45 [95% CI: 0.26-0.76]). ShallowHRDv2 is a performant, clinically validated, and cost-effective test for HRD detection.

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Results of Lung Transplantation for Cystic Fibrosis With Selected Donors Over 65 Years Old.

Lung transplantation is limited by the shortage of suitable donors. Many programs have begun to use extended criteria donors. Donors over 65 years old are rarely reported, especially for young cystic fibrosis recipients. This monocentric study was conducted for cystic fibrosis recipients from January 2005 to December 2019, comparing two cohorts according to lung donor age (<65 years or ≥65 years). The primary objective was to assess the survival rate at 3 years using a Cox multivariable model. Of the 356 lung recipients, 326 had donors under 65 years, and 30 had donors over 65 years. Donors' characteristics did not differ significantly in terms of sex, time on mechanical ventilation before retrieval, and partial pressure of arterial oxygen/fraction of inspired oxygen ratio. There were no significant differences in post-operative mechanical ventilation duration and incidence of grade 3 primary graft dysfunction between the two groups. At 1, 3, and 5 years, the percentage of predicted forced expiratory volume in 1 s (p = 0.767) and survival rate did not differ between groups (p = 0.924). The use of lungs from donors over 65 years for cystic fibrosis recipients allows extension of the donor pool without compromising results. Longer follow-up is needed to assess the long-term effects of this practice.

In Vitro Testing of the Virus-Like Drug Conjugate Belzupacap Sarotalocan (AU-011) on Uveal Melanoma Suggests BAP1-Related Immunostimulatory Capacity.

Purpose: The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity. Methods: We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC50) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq. Results: Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm2) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines. Conclusions: AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC50 and higher CRT exposure.

Benefits of participation in a community of practice focused on evaluation and programmatic improvement for environmental educators.

One of the biggest challenges environmental education (EE) practitioners face is having timely and systematically-collected evaluation data to inform the design and improvement of existing programs. One potential way to provide systematic evaluations of programs and build evaluation capacity for practitioners is through a facilitated community of practice (CoP). We developed a CoP involving 37 organizations who were pivoting to online EE programs within the United States due to the COVID-19 pandemic. Our goals were to build organizational capacity in evaluation and adaptive management to improve these organizations' online EE programs. We describe our CoP design, challenges associated with its implementation, and the benefits reported by participants in the CoP. Participants reported that they improved their evaluation skills and attitudes towards evaluation and developed social capital with a new network of colleagues. They also reported positive changes in practice, both individually and organizationally; considered new outcomes for their programs; and learned about using evaluation data to systematically improve programs. Educators shared their learning both within and outside of their organizations. Those who were more regularly involved in this community reported more positive benefits than others who were less involved. We share our reflections on the process and make suggestions for other evaluators to consider in similar CoP designs.

FAK Inhibitor-Based Combinations with MEK or PKC Inhibitors Trigger Synergistic Antitumor Effects in Uveal Melanoma.

Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers.

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

Circulating Tumor DNA as a Prognostic Factor in Patients With Resectable Hepatic Metastases of Uveal Melanoma.

OBJECTIVE: We report here the results of a prospective study of circulating tumor DNA (ctDNA) detection in patients undergoing uveal melanoma (UM) liver metastases resection (NCT02849145). BACKGROUND: In UM patients, the liver is the most common and often only site of metastases. Local treatments of liver metastases, such as surgical resection, have a likely benefit in selected patients. METHODS: Upon enrollment, metastatic UM patients eligible for curative liver surgery had plasma samples collected before and after surgery. GNAQ / GNA11 mutations were identified in archived tumor tissue and used to quantify ctDNA by droplet digital polymerase chain reaction which was then associated with the patient's surgical outcomes. RESULTS: Forty-seven patients were included. Liver surgery was associated with a major increase of cell-free circulating DNA levels, with a peak 2 days after surgery (∼20-fold). Among 40 evaluable patients, 14 (35%) had detectable ctDNA before surgery, with a median allelic frequency of 1.1%. These patients experienced statistically shorter relapse-free survival (RFS) versus patients with no detectable ctDNA before surgery (median RFS: 5.5 vs 12.2 months; hazard ratio=2.23, 95% CI: 1.06-4.69, P =0.04), and had a numerically shorter overall survival (OS) (median OS: 27.0 vs 42.3 months). ctDNA positivity at postsurgery time points was also associated with RFS and OS. CONCLUSIONS: This study is the first to report ctDNA detection rate and prognostic impact in UM patients eligible for surgical resection of their liver metastases. If confirmed by further studies in this setting, this noninvasive biomarker could inform treatment decisions in UM patients with liver metastases.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.